A CRITICAL STUDY ON ENZYMES
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 Enzyme
Enzymes are large biological molecules responsible for thousands of chemical inter-conversions that sustain life (Smith, 1997). All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds, they are denatured at high temperature and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000 units. Enzymes do not cause reactions to take place, but rather they enhance the rate of reactions that would have been slower without their presence and still remains unused and unchanged.
Many enzymes require the presence of other compounds - cofactors - before their catalytic activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e. apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ionactivator) is called the holoenzyme (Alexopoulos et al., 1996)
The living cell is the site of tremendous biochemical activity called metabolism. It is the process of chemical and physical change which goes on continually in the living organism involving the build-up of new tissues, replacement of old tissue, conversion of food to energy, disposal of waste materials, reproduction - all the activities that we characterize as "life."Thephenomenon of enzyme catalysis makes possible biochemical reactions necessary for all life processes. Catalysis is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change. Synthetic molecules called artificial enzymes also display enzyme like catalysis (Grovesm, 1997).
The catalysts of biochemical reactions are enzymes and are responsible for bringing about almost all of the chemical reactions in living organisms. Without enzymes, these reactions take place at a rate far too slow for the pace of metabolism(Bairoch, 2000).
Enzymes actually work by lowering the activation energy of a reaction. This is achieved when it creates an alternative pathway which is faster for the reaction hence speeding it up such that products are formed faster. Enzyme catalysed reactions are million times faster than uncatalysed reactions, they alter the rates but not the equilibrium constant of the reaction being catalysed (Ashokkumar et al., 2001). A few RNA molecules called ribozymes also catalyse reactions, with an important example being some parts of ribosome (Lilley, 2005).
1.1.1 Types of enzymes
Metabolic enzymes: These have been called the spark of life, the energy of life and the vitality of life. These descriptions are not without merit. Metabolic enzymes catalyse and regulate every biochemical reaction that occurs within the human body, making them essential to cellular function and health (Sangeethaet al.,2005). Digestive enzymes turn the food we eat into energy and unlock this energy for use in the body. Our bodies naturally produce both digestive and metabolic enzymes as they are needed. They either speed up or slow down the chemical reactions within the cells for detoxification and energy production. The enable us to see, hear, and move and think. Every organ, every tissue and all 100 trillion cells in our body depend upon the reaction of metabolicenzymes and enjoy their energy factor. Without these metabolic enzymes, cellular life would beimpossible.
Food enzymes:These are introduced to the body through the raw foods we eat and throughconsumption of supplemental enzyme products. Raw foods naturally contain enzymes providing asource of digestive enzymes when ingested(Hossainet al., 1984). However, raw food manifests only enough enzymesto digest that particular food, not enough to be stored in the body for later use (the exceptionsbeing pineapple and papaya, the sources of the enzymes bromelain and papain). The cooking andprocessing of food destroys all of its enzymes. Since most of the foods we eat are cooked orprocessed in some way and since the raw foods we do eat contain only enough enzymes toprocess that particular food (Persike et al., 2002) our bodies must produce the majority of the digestive enzymes werequire, unless we use supplemental enzymes to aid in the digestive process. A variety ofsupplemental enzymes are available through different sources. It is important to understand thedifferences between the enzyme types and ensure that one is using an enzyme product which willmeet one’s particular needs.
Plant based enzymes:These are the most popular choice of enzymes. They are grown in a laboratorysetting and extracted from Aspergillus species. The enzymes harvested from Aspergillusspecies are called plantbased, microbial and fungal. Of all the choices, plant based enzymes are the most active. Thismeans they can break down more fat, protein and carbohydrates in the broadest pH range than any other sources (Ashokkumar et al., 2001).
1.1.2 Characteristics of enzymes
Protein nature:Enzyme is a protein. The main components of an enzyme is protein.
Temperature:Enzymes are sensitive to temperature. Many work best at temperatures close to body temperatures and most lose their ability to catalyse if they are heated above 60 or 70o C. (Ashokkumar et al., 2001).
Acidity and alkalinity:Many enzymes work best at a particular pH and stop working if the pH becomes too acidic or alkaline.
Catalytic effect:It acts as catalyst, enzyme functions in accelerating chemical reaction, but the enzyme itself does not change after the reaction ends.
Specificity:It functions specifically. The enzyme only catalyzes one kind of substrate and cannot function for many substrates. The term is called one enzyme one substrate.
Reversibility: It means the enzyme does not determine the direction of reaction, but it only functions in accelerating reaction rate until it reaches equilibrium. The enzyme also functions in substance synthesis and substance breaking down reaction.
Small quantity:It is required, in small amount. A small amount of enzyme is able to catalyze a chemical reaction (Nason, 1968).
Table 1: Classification of enzyme by nature and the reaction they catalyse
Classification Type of Reaction Catalysed
Oxidoreductases Oxidation-reduction reactions
Transferases Transfer of functional groups
Hydrolases Hydrolysis reactions
Lyases Group elimination to form double bonds
Isomerases Isomerization
Ligases Bond formation coupled with ATP
hydrolysis
1.1.3 Classes of enzymes
Oxidoreductase: These enzymes catalyze redox reactions, i.e., reactions involving the transfer of electrons from one molecule to another. In biological systems we often see the removal of hydrogen atoms from a substrate. Typical enzymes catalyzing such reactions are called dehydrogenases. For example, alcohol dehydrogenase catalyzes reactions of the type R-CH2OH + A → R-CHO + AH2, where A is a hydrogen acceptor molecule. Other examples of oxidoreductases are oxidases and laccases, both catalyzing the oxidation of various substrates by dioxygen, and peroxidases, catalyzing oxidations by hydrogen peroxide. Catalases are a special type, catalyzing the disproportionation reaction 2H2O2 → O2 + 2H2O, whereby hydrogen peroxide is both oxidized and reduced at the same time. (Alexopoulus et al., 1996)
Transferases: Enzymes in this class catalyze the transfer of groups of atoms from one molecule to another or from one position in a molecule to other positions in the same molecule. Common types are acyltransferases and glycosyltransferases. CGTase (cyclodextrin glycosyltransferase) is one such enzyme type, which moves glucose residues within polysaccharide chains in a reaction that forms cyclic glucose oligomers (cyclodextrins).
Hydrolases: Hydrolases catalyze hydrolysis, the cleavage of substrates by water. The reactions include the cleavage of peptide bonds in proteins by proteases, glycosidic bonds in carbohydrates by a variety of carbohydrases, and ester bonds in lipids by lipases. In general, larger molecules are broken down to smaller fragments by hydrolases (Alexopoulus et al., 1996)
Lyases:Lyases catalyze the addition of groups to double bonds or the formation of double bonds though the removal of groups. Thus bonds are cleaved by a mechanism different from hydrolysis. Pectate lyases, for example, split the glycosidic linkages in pectin in an elimination reaction leaving a glucuronic acid residue with a double bond.
Isomerases: Isomerases catalyze rearrangements of atoms within the same molecule; e.g., glucose isomerase will convert glucose to fructose.
Ligases: Ligases join molecules together with covalent bonds in biosynthetic reactions. Such reactions require the input of energy by the concurrent hydrolysis of a diphosphate bond in ATP, a fact which makes this kind of enzyme difficult to apply commercially.
1.2 industrial applications of enzymes
The catalytic ability of enzymes has made it of utmost importance in various industry such as food industries, pharmaceutical industries, Health sector, agricultural sectors, clothing and textile etc. Enzymes found in nature have been used since ancient times in the production of food products, such as cheese, sourdough, beer, wine and vinegar, and in the manufacture of commodities such as leather, indigo and linen and processes relied on either enzymes produced by spontaneously growing microorganisms or enzymes present in added preparations such as calves’ rumen or papaya fruit although the enzymes were, accordingly, not used in any pure or well-characterized form.
In the last century, several developments has made it possible to manufacture enzymes in purified and well-characterized preparations and even on a large scale. This developments allowed the introduction of enzymes into true industrial products and processes, for example, within the detergent, textile and starch industries. The use of recombinant gene technology also has further improved manufacturing processes and enabled the commercialization of enzymes that could previously not be produced.
Latest developments within modern biotechnology i.e. the introduction of protein engineering and directed evolution, have further revolutionized the development of industrial enzymes. These advances have made it possible to provide tailor-made enzymes displaying new activities and adapted to new process conditions, enabling a further expansion of their industrial use.
Wine manufacturing: Much of the early interest in enzymology was developed by scientists like Pasteur, Payen and Persoz, who were associated with food, wine, and beer industries. Pasteur was perhaps best known to the French nation as the “saviour of the wine industry” because his pasteurization process salvaged an ailing industry beset with problems of microbial contamination. Papain is used in brewing industry as a stabilizer for chill-proof beer, because it removes small amounts of protein that cause turbidity in chilled beer (Shafiq et al., 2002).
Cheese making: Since long the animal rennin (or rennet) is employed in making cheese. The enzyme rennet is obtained on a commercial scale from the fourth or true stomach of the unweaned calves which are specifically slaughtered for this purpose. One calf produces only 5 to 10 gm. of rennet. The enzyme helps in coagulating the casein of milk. Certain preservatives (boric acid, benzoic acid or sodium chloride) are, sometimes, added to prevent decomposition of the enzyme preparations by bacteria. An enzyme lipase is added to cheese for imparting flavor to it. Many vegetarians are unaware that the cheese made in India contains animal rennet. However, an international charitable trust concerned with the welfare of animals, the Beauty Without Cruelty (BWC) has, with the help of Aurey Dairy, Mumbai, undertaken successful experimental trials in cheese making using non animal rennet .(Stanieret al., 1970).
Candy making: An enzyme, invertase helps preventing granulation of sugars in soft-centred candies. Another enzyme, lactase prevents formation of lactose crystals in ice cream which would otherwise not allow the product seem sandy in texture.
Bread whitening: Lipoxygenase is used for whitening the bread.
Clarifying fruit-juices: The enzymes are being used in processing of fruit juices such as apple juice and grape juice. The juices are clarified by adding a mixture of pectic enzymes which hydrolyze the pectic substances causing turbidity.
Tenderizing meat: Because hydroxyprolyl residues create bends in collagen helices, which contribute to the tough and rubbery texture often associated with cooked meat, treating the meat with a protease (bromelain or papain) prior to its cooking hydrolyzes peptide bonds, and thus tenderizes it. (Schell et al., 2002)
Desizing fabrics: The woven fabrics are sized by applying starch to the warp (lengthwise) threads to strengthen the yarn before weaving. But when these fabrics are printed or dyed, the sizing should be removed. Desizing may be done by acids, alkalis’ or enzymes. Enzymatic desizing is, however, preferred as it does not weaken the fabrics. Enzymes for this purpose are obtained from a variety of sources including bacteria, fungi and malt.
Destaining fabrics: In dry-cleaning, the stains due to glue, gelatin or starch are removed by employing certain enzymes, such as alcalase.
Dehairing hide: In the manufacture of leather, the hide is made free from hair. This is done by employing pancreatic enzymes which hydrolyze the proteins of the hair follicles, thus freeing the hair so that it may be easily scraped off from the hide.
Recovering silver: Pepsin is used to digest gelatin in the process of recovering silver from photographic films (Wang, 1998).
.Correcting digestion: When the enzymes are present insufficiently in the body, certain digestive disorders come up. These may be cured by supplying the lacking enzymes. Pepsin, papain and amylases aid digestion in the stomach while pancreatic enzymes act in the duodenum.
Wound healing: Proteolytic enzymes from pig pancreas are used to alleviate skin diseases, bed sores and sloughing wounds. These enzymes act by destroying proteolytic enzymes of man, that prevent the healing of such wounds. The enzymes commonly used for wound debridement are the proteases such as streptodornase, ficin and trypsin.
Analyzing biochemical: Certain enzymes are used in clinical analysis. For example, uricase and urease are employed in the determination of uric acid and urea respectively in blood.Besides, sucrose and raffinose contents in sugar mixtures are determined by polarimetrybefore and after treating the solutions with the enzymes, sucrase and melibiase.
Dissolving blood clot: The enzyme urokinase, which is manufactured from urine, is being used effectively in Japan in the treatment of blood clot in brain, artery and other circulatory diseases. A team of Soviet scientists led by Yevgeni Chazov, Director of the National Cardiological Research Centre, Moscow, have, in 1982, developed an effective enzyme streptodekase, which can dissolve blood clots in vessels. The new enzyme is particularly useful in preventing heart attacks as clots are responsible for 9 out of 10 fatal cases of cardiac arrests (Hansen, 1990).
Changing the blood type: Scientists have successfully employed several types of specific enzymes in an epoch-making experiment to freely change human blood types. They found that the composition of polysaccharide on the surface of blood corpuscles determines each person’s type of blood. Different kinds of sugar characteristics of each blood type form on the surface of RBCs due to the function of a synthetic enzyme. If the sugar is separated from the surface of RBCs by using a specific decomposition enzyme, type A blood and type B blood can be reverted to type O, the prototype of the two blood types. If this breakthrough can be put to practical use, it will fulfil a long-cherished dream of doctors to administer blood transfusions irrespective of the type of blood a patient has by merely changing the patient’s blood type to match the blood available (Koshland, 1984).
Diagnosing hypertension: A new method called radio immunoassay procedure for diagnosing cases of hypertension has been developed by Bhabha Atomic Research Centre (BARC). In it, the activity of renin, a proteolytic enzyme secreted by the kidneys, is calculated indirectly by measuring angiotensin-I which is formed by the action of renin. Renin acts as part of a complex feedback mechanism for regulating blood volume and pressure (Ashokkumar et al., 2001).
Augmenting surgery: A technique using the enzyme trypsin as an adjunct to cataract surgery has been developed. With older techniques, it required an incision about 2.5 cm long in the white of the eye to remove the clouded lens. Modern microsurgery has, however, reduced this cut to only 0.3 cm. But the new method involves a still smaller cut wide enough for a needle 0.025 cm wide. The hollow needle is used to inject a microscopic amount of trypsin, a digestive enzyme secreted by the pancreas. Trypsin digests and liquefies the semisolid interior of the lens without harming other parts of the eye. Once the enzyme liquefies the lens—which takes from a few hours to overnight—the lens is removed by suction through the same hollow needle. This eliminates the necessity of intervention in the eye, the constant passing in and out of the instruments and suturing. The lesser the tissue is wounded, the quicker it recovers. This enzyme surgery for cataracts could be done as an outpatient operation. The patients would come in one day to have the enzyme injected and return the next day to have the cataract removed (Dahot and Noomrio, 1996).
Breaking down chemicals: Recently, in 1993, a group of scientists from the Netherlands led by Han G. Brunner have found a tiny genetic defect that appears to predispose some men toward aggression, impulsiveness and violence. The afflicted persons often react to the mostmildly stressful occasions with aggressive outbursts, cursing or assaulting the persons they deem a threat. The researchers have linked the abnormal behaviours to mutations in the gene responsible for the body’s production of monamine oxidase-a, an enzyme critical for breaking down chemicals that allow brain cells to communicate. It is proposed that lacking the enzyme, the brains of afflicted men end up with excess deposits of potential signalling molecules like serotonin, dopamine and noradrenaline. Those surplus neurotransmitters, in turn, stimulate often hostile conduct. The erratic behaviour is due to point mutation. The gene is on the X chromosome, which explains why only males, with their single copy of the X chromosome, can suffer from the enzyme deficiency. Women can serve as carriers of the genetic defect, but are themselves protected from its symptoms by their possession of a second, good copy of the gene, sitting on their second X chromosome. Although the number of persons afflicted with this disease is not known but based on other types of hereditary disorders, the researchers estimate that the illness is likely to be quite rare in the general population, i.e., no more than one in 100,000 people (Benkebliaet al., 2007)
Destroying acids: Sprouts are the natural health boosters. They are basically the young new plants and are, in fact, the organic answer to simple natural health. Almost any edible seed can be sprouted. Far from being the invention of food faddists, sprouting dates back to 2939 B.C. in China. Sprouts are found in all shapes and colours and it is best to choose these from legume plants. Sprouting greatly improves the safety and nutritional quality of all pulses, seeds and grains. The enzymes which go into action during sprouting not only neutralize trypsin-inhibiting factors but also destroy harmful acids like phytic acid. Phytic acid, an integral constituent of grains, tends to bind minerals, making them unavailable to the body.
Syrup manufacturing: An immobilized enzyme is one that is physically entrapped or covalently-bonded by chemical means to an insoluble matrix, e.g., glass beads, polyacrylamide or cellulose. Immobilization of an enzyme often greatly enhances its stability, which makes its prolonged catalytic life a valuable industrial trait (Kotwal and Shankar, 2009). These days, immobilized glucoseisomerase is being successfully used in the production of high-fructose corn syrup, esp., in the United States.
1.3Fungi as a micro-organism.
Fungi are heterotrophic organisms belonging to the eukaryotic kingdom. Occurring worldwide, there are some 70,000 species of fungi including mushrooms, moulds, rusts, smuts, truffles, puffballs, morels and yeasts (Alexopoulos et al., 1996).
About 80 000 to 120 000 species of fungi have been described to date, although the total number of species is estimated at around 1.5 million (Papagianni, 2004). This would render fungi one of the least-explored biodiversity resources of our planet. It is notoriously difficult to delimit fungi as a group against other eukaryotes, and debates over the inclusion or exclusion of certain groups have been going on for well over a century. In recent years, the main arguments have been between taxonomists striving towards a phylogenetic definition based especially on the similarity of relevant DNA sequences, and others who take a biological approach to the subject and regard fungi as organisms sharing all or many key ecological or physiological characteristics-the ‘union of fungi’ (Moore-Landecker, 1996). Being interested mainly in the way fungi function in nature and in the laboratory, we take the latter approach and include several groups in this book which are now known to have arisen independently of the monophyletic ‘true fungi’ (Eumycota) and have been placed outside them in recent classification schemes
Based on their lifestyle, fungi may be circumscribed by the following set of characteristics(Moore-Landecker, 1996):
Nutrition: Heterotrophic (lacking photosynthesis), feeding by absorption rather than ingestion.
Vegetative state: On or in the substratum, typically as a non-motile mycelium of hyphae showing internal protoplasmic streaming. Motile reproductive states may occur (St-Germain and Summerbell ,1996).
Cell wall: Typically present, usually based on glucans and chitin, rarely on glucans and cellulose (Oomycota).
Nuclear status: Eukaryotic, uni- or multinucleate, the thallus being homo- or heterokaryotic, haploid, dikaryotic or diploid, the latter usually of short duration (but exceptions are known from several taxonomic groups).
Life cycle: Simple or, more usually, complex.
Reproduction: The following reproductive events may occur: sexual (i.e. nuclear fusion and meiosis) and/or parasexual (i.e. involving nuclear fusion followed by gradual de-diploidization) and/or asexual (i.e. purely mitotic nuclear division).
Propagules: These are typically microscopically small spores produced in high numbers.
Motile spores are confined to certain groups.
Sporocarps: Microscopic or macroscopic and showing characteristic shapes but only limited tissue differentiation.
Habitat: Ubiquitous in terrestrial and freshwater habitats, less so in the marine environment.
Ecology: Important ecological roles as saprotrophs, mutualistic symbionts, parasites, or hyperparasites.
Distribution: Cosmopolitan
1.3.1 How fungi reproduce
Fungi are capable of both sexual and asexual reproduction.When a fungus reproduces sexually it forms adiploid zygote, as do animals and plants. Unlike animalsand plants, all fungal nuclei except for the zygote are haploid,and there are many haploid nuclei in the commoncytoplasm of a fungal mycelium (Nguyenet al., 2005). When fungi reproducesexually, hyphae of two genetically different mating typescome together and fuse (Nakano et al., 2000). In two of the three phyla offungi, the genetically different nuclei that are associatedin a common cytoplasm after fusion do not combine immediately.Instead, the two types of nuclei coexist formost of the life of the fungus. A fungal hypha containingnuclei derived from two genetically distinct individuals iscalled a heterokaryotic hypha. If all of the nuclei are geneticallysimilar to one another, the hypha is said to behomokaryotic. If there are two distinct nuclei withineach compartment of the hyphae, they are dikaryotic. Ifeach compartment has only a single nucleus, it ismonokaryotic. Dikaryotic hyphae have some of the geneticproperties of diploids, because both genomes aretranscribed. These distinctions are important in understandingthe life cycles of the individual groups (Kumar and Jam, 2003).
Cytoplasm in fungal hyphae normally flows throughperforated septa or moves freely in their absence. Reproductivestructures are an important exception to this generalpattern. When reproductive structures form, they arecut off by complete septa that lack perforations or haveperforations that soon become blocked. Three kinds of reproductivestructures occur in fungi:
Sporangia: whichare involved in the formation of spores
Gametangia:structures within which gametes form; and
Conidiophores:structures that produce conidia, multinucleateasexual spores.Spores are a common means of reproduction amongfungi. They may form as a result of either asexual or sexualprocesses and are always non-motile, being dispersed bywind. When spores land in a suitable place, they germinate,giving rise to a new fungal hypha. Because the spores arevery small, they can remain suspended in the air for longperiods of time. Because of this, fungal spores may beblown great distances from their place of origin, a factor inthe extremely wide distributions of many kinds of fungi.Unfortunately, many of the fungi that cause diseases inplants and animals are spread rapidly and widely by suchmeans. The spores of other fungi are routinely dispersed byinsects and other small animals. (Alexopoulos et al., 1996).
1.3.2 Growth and physiology of fungi
The growth of fungi as hyphae on or in solid substrates or as single cells in aquatic environments is adapted for the efficient extraction of nutrients, because these growth forms have high surface area to volume ratios (Hayden and Maude 1994). Hyphae are specifically adapted for growth on solid surfaces, and to invade substrates and tissues.They can exert large penetrative mechanical forces; for example, the plant pathogenMagnaporthe grisea forms a structure called an appressorium that evolved to puncture plant tissues. The pressure generated by the appressorium, directed against the plant epidermis, can exceed 8 megapascals (1,200 psi) (Mammaet al.,2008). The filamentous fungus Paecilomyces lilacinus uses a similar structure to penetrate the eggs of nematodes.
The mechanical pressure exerted by the appressorium is generated from physiological processes that increase intracellular turgor by producing osmolytes such as glycerol (Tafintaet al., 2013).Adaptations such as these are complemented by hydrolytic enzymes secreted into the environment to digest large organic molecules—such as polysaccharides, proteins, and lipids—into smaller molecules that may then be absorbed as nutrients (Pereiraet al.,2007; Schalleret al.,2007). The vast majority of filamentous fungi grow in a polar fashion—i.e., by extension into one direction—by elongation at the tip (apex) of the hypha. Other forms of fungal growth include intercalary extension (longitudinal expansion of hyphal compartments that are below the apex) as in the case of some endophytic fungi(Hayden and Maude, 1994)or growth by volume expansion during the development of mushroom stipes and other large organs. Growth of fungi as multicellular structures consisting of somatic and reproductive cells—a feature independently evolved in animals and plants—has several functions, including the development of fruit bodies for dissemination of sexual spores (see above) and biofilms for substrate colonization and intercellular communication (Collier et al., 1998).
The fungi are traditionally considered heterotrophs, organisms that rely solely on carbon fixed by other organisms for metabolism(Vaija and Linko, 1986). Fungi have evolved a high degree of metabolic versatility that allows them to use a diverse range of organic substrates for growth, including simple compounds such as nitrate, ammonia, acetate, or ethanol(Marzlufet al., 1981; Heyneset al., 1994). In some species the pigment melanin may play a role in extracting energy from ionizing radiation, such as gamma radiation. This form of "radiotrophic" growth has been described for only a few species, the effects on growth rates are small, and the underlying biophysical and biochemical processes are not well known. This process might bear similarity to CO2 fixation via visible light, but instead using ionizing radiation as a source of energy (Hayden and Maude, 1994).
1.4. Production of enzyme by fermentation technologies
Fermentation technology is a field which involves the use of microorganisms and enzymes for production of compounds which have application in the energy, material, pharmaceutical, chemical and the food industry. Though fermentation processes are used for generations for the requirement for sustainable production of materials and energy is demanding creation and advancement of novel fermentation processes. Efforts are directed both to the advancement of cell factories and enzymes as well as of design of new processes, concepts and technologies for fermentation process. Through fermentation, we can produce enzymes for industrial purposes. Process of Fermentation includes the use of microorganisms, like yeast and bacteria for the production of enzymes. Mainly, there are two methods of fermentation which are used to produce enzymes. First is submerged fermentation and second is solid-state fermentation.
1.4.1. Solid-State Fermentation (SSF)
SSF utilizes solid substrates, like bran, bagasse, and paper pulp(Arandaet al., 2006). The main advantage of using these substrates is that nutrient-rich waste materials can be easily recycled as substrates(Gutierrez-Correaet al., 1999). In this fermentation technique, the substrates are utilized very slowly and steadily, so the same substrate can be used for long fermentation periods. Hence, this technique supports controlled release of nutrients. SSF is best suited for fermentation techniques involving fungi and microorganisms that require less moisture content (Kubicek and Röhr, 1989). However, it cannot be used in fermentation processes involving organisms that require high Aw (water activity), such as bacteria. (Ashokkumar et al., 2001)
1.4.2. Submerged Fermentation (SmF)/Liquid Fermentation (LF)
SmF utilizes free flowing liquid substrates, such as molasses and broths. The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients. This fermentation technique is best suited for microorganisms such as bacteria that require high moisture content(Leangon and Maddox, 2000). An additional advantage of this technique is that purification of products is easier. SmF is primarily used in the extraction of secondary metabolites that need to be used in liquid form (Ashokkumar et al., 2001).
1.4.3. Substrates used for fermentation
The outcome of fermentation highly varies for each substrate; hence, it is extremely important to choose the right substrate. Fermentation techniques have to be optimized for each substrate. This is primarily due to the reason that an organism reacts differently to each substrate. The rates of utilization of various nutrients differ in each substrate, and so does productivity(Andjelkovicet al., 2010). Some of the common substrates used in solid state fermentation are wheat bran, rice and rice straw, hay, fruit and vegetable waste, paper pulp, bagasse, coconut coir, and synthetic media (Kernet al., 1992). Some common substrates used in submerged fermentation are soluble sugars, molasses, liquid media, fruit and vegetable juices, and sewage/waste water (Ngadi and Correia, 1992).
1.5. INVERTASE
Invertase is an enzyme that catalyzes the hydrolysis (breakdown) of sucrose (table sugar) into its component parts, glucose and fructose (Vitolo et al., 1995),The resulting mixture of fructose and glucose is called inverted sugar syrup (Liuet al., 2006). Related to invertases are sucrases. Invertases and sucrases hydrolyze sucrose to give the same mixture of glucose and fructose(Akeshige and Ouchi, 1995). Invertases cleave the O-C(fructose) bond, whereas the sucrases cleave the O-C(glucose) bond (Vitoloet al 1995). Invertases are produced by bacteria, fungi, higher plants and some animal cells (Matrai et al., 2000; Huang et al., 2003).
It is generally derived from a beneficial strain of Saccharomyces cerevisiae and then purified to be used either by itself or as a part of a multi-enzyme formula(Vitolo et al., 1995). Combined with other carbohydrates, it enhances the overall digestion of starch, sugar and other carbohydrates. Invertases’ ability to break down (hydrolyze) the bond between fructose and glucose makes it a vital part of the digestion of complex sugars into blood sugar (glucose) which can be used as a ready fuel source by the body(Aricaet al., 2000). It is also known as beta-fructofuranosidase and may be listed under this name on some product labels and scientific literature (Sanjay and Sugunan, 2006). Invertase is one of the essential enzymes nature uses to help us digest sugars. Commonly found in bee pollen and yeast sources, invertase plays a key role not only in digestive processes(Belcarz et al., 2002), but also, and perhaps more importantly, in overall human disease prevention, physical rejuvenation and anti-ageing processes. As we age, we have less access to this natural enzyme, resulting in a reduced ability to extract the vital nutrients from the food we eat. It can also slow our digestive process, as sugars and starches are such a big part of most American diets (Stommel et al., 1985). And, while some forms of sugar and carbohydrates are good for the body, they cannot be absorbed or digested well without the help of the invertase enzyme (Fernandes and Benda 1985).
It is also created naturally by bees, who use its ability to hydrolyze the sugars in raw nectar to create the delicious honey that we eat (Hussainet al., 2009). Honeybees produce massive amounts of the enzyme as it breaks the bond between glucose and fructose, adding hydrogen and hydroxide(Rashad and Nooman, 2009). In contrast to many other enzymes, invertase has the distinct ability to remain active within a wide range of pH levels.For industrial use, invertase is usually derived from yeast. It is also synthesized by bees, who use it to make honey from nectar. Optimum temperature at which the rate of reaction is at its greatest is 60 °C and an optimum pH of 4.5 (Benkebliaet al., 2007). Typically, sugar is inverted with sulfuric acid.
Invertase is extensively used in confectilonaries, food industries and in pharmaceuticals (Ashokkumar et al., 2001). Microbial invertase is used for the manufacture of calf food and food for honey bees. Many organisms produce invertase such as Neurospora crassa, Candida utilis, Fusarium oxyspor Phytophthora meganosperma, Aspergillus niger, Saccharomyces cerevisiae, Schizosaccharomyces pombe andSchwanniomyces occidentalis (Silveira et al., 2000). Saccharomyces cerevisiae is the organism of choice for invertase production because of its characteristic high sucrose fermentability.
1.5.1. Benefits of Invertase
Natural Immune Booster
Enzymes found in honey, such as invertase have been studied for their metabolic activity. Studies done on asparagus (Asparagus officinalis) found that high invertase activity found in the top portion of asparagus spears might be related to the high metabolism occurring in this portion (Benkeblia et al., 2007).
Antioxidant Support
Invertase has many antioxidant properties, and it is a powerful agent against harmful organisms. These two aspects allow it to aid in the defense against of bacterial infestations and gut fermentation due to oxidation. In Ancient India, raw honey was often used in patients with a weak heart. It was known to kill off bacteria and reduce intestinal ailments. It was also used for its hygroscopic (moisture-retaining) properties, and its ability to pull moisture out of the body, causing bacterial infestations to subside. Invertase is one of these key elements of the enzymatic support found in honey (Xuet al., 2003)
Ulcers
Because invertase creates pre-digested simple sugars, it helps reduce stomach toxicity, in that sugars do not remain in the stomach long enough to create toxic fermentation. Fermentation is what causes bacteria and disease to build up in the digestive tract. In this way, invertase helps protect the body from ulcers, as well as many other digestive diseases (Vitoloet al., 1995).
Naturally Toxic to Harmful Organisms
Again, in honey, enzymes such as invertase show the ability to turn glucose into natural hydrogen peroxide (Benkeblia et al., 2007).
Natural Respiratory Support
Enzymes including invertase have been shown to help reduce colds, flu and other respiratory infections. One European study on 18,000 patients found that honey drastically helped upper respiratory tract infections such as bronchitis, asthma and allergies(Benkeblia et al., 2007).
Cancer Support
Some medicinal studies also show that the invertase enzyme may exhibit some chemotherapeutic properties (Rubio et al., 2002). Research done in Australia and Japan have found that the enzymes in honey helped support patients with advanced cases of both bone and stomach cancer. In some cases, the cancers even went into regression. Currently, enzyme therapy is being used as a vital component of many natural cancer therapies. European researchers reported, “Studies showed that enzyme therapy can reduce the adverse effects caused by radiotherapy and chemotherapy. There is also evidence that, in some types of tumours, survival may be prolonged and that the beneficial effect of systemic enzyme therapy seems to be based on its potential to reduce redness and swelling (Hubertet al., 2007)
The product VeganZyme™ contains a 100% vegan form of Invertase produced by the natural fermentation process of Saccharomyces cerevisae. It comes from all vegetarian, non-GMO sources, is kosher certified, gluten free, contains no animal product and is completely suitable for vegetarians and vegans (Bayramogluet al., 2003).
VeganZyme™ is the most advanced full-spectrum systemic and digestive enzyme formula in the world and is free from fillers and toxic compounds. This formula contains digestive enzymes which help digest fats (lipids), sugars, proteins, carbohydrates, gluten, fruits and vegetables, cereals, legumes, bran, nuts and seeds, soy, dairy and all other food sources. VeganZyme™ may also be used as a systemic enzyme blend to break down excess mucus, fibrin, various toxins, allergens, as well as excess clotting factors throughout your body.
1.5.2.Measurement of invertase activity:
For invertase activity, 2.5 ml acetate buffer(50 mM, pH 5.5) and 0.1 ml sucrose (300mM) was added into the individual testtubes. The tubes were pre-incubated at35°C for 5 min. After the addition of 0.1 mlof appropriately diluted enzyme solution,incubation was continued for another 5min. The reaction mixture was placed in aboiling water bath for 5 min., to stop thereaction and allowed to cool at roomtemperature. A blank was also run parallelreplacing the enzyme solution with distilledwater. To 1.0 ml of each reaction mixture1.0 ml of DNS was added and the tubesplaced in boiling water for 5 min. Aftercooling to 20ºC, volume was raised up to10 ml. Transmittance was measured at 546nm using spectrophotometer (Guimaraes et al., 2007)
1.6. Objective
The objectives of this study were to:
1. isolate fungal species from decaying orange fruits collected from the Obafemi Awolowo University new market;
2. characterize the fungal isolates and
3. screen the fungal isolates for their ability to produce invertase.
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